PROJECT SUMMARY The voltage-gated sodium channel (Na-channel) is a central component of cardiac electrogenesis. Its dysfunction can lead to arrhythmias that cause sudden cardiac death both in acquired (ischemia; heart failure), and genetic disorders. Rare and common genetic variants in SCN5A, encoding the pore-forming Na-channel subunit NaV1.5, are strongly associated with life-threatening arrhythmias and with structural heart disease. Yet, insight into Na-channel function beyond electrogenesis, is limited. Na-channels form macromolecular complexes that, in adult ventricular myocytes, cluster at specific subcellular domains (or ?pools?). A key emerging concept is that certain Na-channel partners localize only to one subcellular domain, endowing the functional complex with region-specific properties that, if disrupted, facilitate arrhythmias. Here, we propose the existence of a major Na-channel subpopulation that clusters with subjacent mitochondria to create an anatomical substrate for electro-metabolic coupling. We further propose that in this subdomain regulation is reciprocal: mitochondria regulate Na-channels and Na-channels regulate mitochondrial function. As such, this hub can be a key node in the genesis of cardiomyopathies associated with Na-channel variants. Based on published as well as preliminary data we propose the following SPECIFIC AIMS: Aim 1. To characterize the molecular composition and function of the Na-channel-mitochondrial hub. Hypothesis: We postulate that the mitochondrial reticulum in the adult cardiac myocyte presents distinct, functionally active hubs that contact the cell membrane at positions enriched for Na-channel proteins. We propose that at these hubs, Na-channel composition/activity can affect mitochondrial structure and function. Aim 2. To define the molecular network that constitutes the SCN5A/NaV1.5 interactome. Hypothesis: We postulate that proteomic approaches will lead to the discovery of yet-unidentified molecules that are a part of the Na-channel/mitochondrial hub, and that knowledge on their identity and nanoscale localization will give new information as to the function of this hub in health and disease. We further speculate that expression and sequence integrity of SCN5A is coupled, directly or indirectly, to a transcriptional regulatory network that modulates cell energy and metabolism. This proposal advances the concept that electro-metabolic coupling is a two-way process where electrical and metabolic activity reach a mutual equilibrium. Successful accomplishment of these experiments will amend basic concepts that are fundamental to our understanding of the electrical and metabolic cell homeostasis.